Monoclonal and oligoclonal anti-egfr antibodies for use in the treatment of tumors expressing predominantly high affinity egfr ligands or tumors expressing predominantly low affinity egfr ligands

ABSTRACT

Disclosed are pharmaceutical preparations for, and methods for determining, appropriate and effective treatment with therapeutic agents comprising a single species of anti-EGFR monoclonal antibody or therapeutic agents comprising a plurality of species of such antibodies, as well as kits useful for making such determinations.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/488,270, filed on Jun. 4, 2012, which claims the benefit of U.S.Provisional Application No. 61/493,252, filed on Jun. 3, 2011, U.S.Provisional Application No. 61/504,633, filed on Jul. 5, 2011, and U.S.Provisional Application No. 61/558,945, filed on Nov. 11, 2011, theentire contents of which are incorporated by reference herein.

BACKGROUND

The epidermal growth factor receptor (EGFR) is a cell surfacetransmembrane receptor of the HER/ErbB receptor family that transmitssignals (including mitogenic signals that drive cell proliferation) tothe interior of a cell when activated, typically by the binding of anyof a number of extracellular ligands such as epidermal growth factor(EGF). EGFR ligands vary in their affinity for EGFR and are categorizedas either high- or low-affinity ligands. It is thought that the high-and low-affinity interactions between EGFR and its ligands activatedifferent signaling pathways. This signal transmission occurs through acascade of intracellular events beginning with protein phosphorylationmediated by receptor tyrosine kinase activity. EGFR has proven aresponsive target for anti-proliferative (e.g., anti-cancer) drugs,including “small molecule” tyrosine kinase inhibitor drugs (typically nolarger than 700-900 AMU) that may be orally administered as well asmonoclonal antibody based drugs that specifically bind to theextracellular domain of EGFR. EGFR-targeted monoclonal antibodies arenot always effective against EGFR-expressing tumors. One approach takenwith the aim of improving anti-EGFR antibody efficacy has been todevelop mixtures of anti-EGFR monoclonal antibodies (i.e., oligoclonalantibodies) targeted to different sites (epitopes) on to theextracellular domain of EGFR. See, e.g., PCT Int. Pub. No.WO/2011/140254 and U.S. Pat. No. 7,887,805. These developments havecreated a need to enable the identification of cancer patients whosetumors have characteristics rendering them unresponsive to monoclonalanti-EGFR antibodies so that such patients may receive effectivetreatment via administration of oligoclonal anti-EGFR antibodies. Thepresent disclosure answers this need and provides other benefits.

SUMMARY

Provided herein are theranostic methods for predicting responsiveness oftumor cells that express EGFR to therapeutic agents comprising anti-EGFRantibodies, and methods based on such predictions for treating patientshaving such tumors with such therapeutic agents. Anti-EGFR antibodiessuch as those monoclonal and oligoclonal antibodies described in PCTInt. Pub. No. WO/2011/140254 and corresponding pending U.S. patentapplication Ser. No. 13/100,920, in pending U.S. provisional patentapplications Nos. 61/504,633 and 61/558,945, and in U.S. Pat. No.7,887,805 (the “Oligoclonal Applications”), as well as oligoclonalmixtures of such antibodies in combination with other anti-EGFRantibodies, are useful for treatment of cancers, e.g., malignant(neoplastic) tumors. Examples of cancers include but are not limited to,carcinoma, adenoma, blastoma, sarcoma, and lymphoma. More particularexamples of such cancers include squamous cell cancer, small-cell lungcancer, non-small cell lung cancer, gastric cancer, pancreatic cancer,glial cell tumors such as glioblastoma and neurofibromatosis, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, melanoma, colorectal cancer, endometrialcarcinoma, salivary gland carcinoma, kidney cancer, renal cancer,prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma andvarious types of head and neck cancer. In a particular embodiment, acancer treated or diagnosed using the methods disclosed herein isselected from melanoma, breast cancer, ovarian cancer, renal carcinoma,gastrointestinal/colon cancer, lung cancer (e.g., NSCLC), and prostatecancer.

Accordingly, theranostic methods predicting which tumors (e.g.,malignant tumors) will be responsive to treatment with oligoclonalanti-EGFR antibodies, but not responsive to treatment with singlemonoclonal anti-EGFR antibodies, are provided. Also provided are kitsuseful for predicting responsiveness of tumors to anti-EGFR monoclonaland oligoclonal antibodies and methods of use thereof.

In one aspect, a preparation comprising only a single species ofanti-EGFR monoclonal antibody (as opposed to preparations comprisingmixtures of monoclonal antibodies) is provided for use in treating apatient having a tumor (e.g., a malignant tumor) that is predicted tohave a favorable outcome (as described below) as a result of treatment#1, i.e., treatment with the preparation comprising only a singlespecies of monoclonal antibody, and as a result of treatment #2, i.e.,treatment with an oligoclonal anti-EGFR antibody preparation comprisinga plurality of species of monoclonal anti-EGFR antibodies, one againsteach of at least two extracellular epitopes of EGFR, one of whichantibody against at least two extracellular epitopes of EGFR being anantibody that inhibits ligand binding to EGFR. In another aspect, apreparation comprising a plurality of species of monoclonal anti-EGFRantibodies is provided for use in treating a patient having a tumor thatis predicted to have a favorable outcome as a result of treatment #2,but not as a result of treatment #1.

Identification of tumors predicted to respond to treatment #1 andtreatment #2, or to treatment #2, but not to treatment #1, comprisesobtaining a biopsy sample of the tumor and:

-   -   a) measuring levels in the sample of at least two (e.g., at        least three or at least four) low affinity EGFR ligands, e.g.,        selected from amphiregulin, epigen, or epiregulin, which may,        for each ligand independently, be measured as levels of ligand        protein or as levels of the corresponding RNA species coding for        the ligand protein, in the biopsy sample,    -   b) measuring levels in the sample of at least two (e.g., at        least three) high affinity EGFR ligands, e.g., selected from        betacellulin, EGF, HB-EGF or TGFα, which may, for each ligand        independently, be measured as protein levels or as levels of the        corresponding RNA species coding for them, in the biopsy sample,        -   in one embodiment, all of the levels measured in a) are            protein levels and all of the levels measured in b) are            protein levels; in another embodiment all of the levels            measured in a) are mRNA levels and all of the levels            measured in b) are mRNA levels.    -   and,    -   c) comparing the average level of each of the high affinity EGFR        ligands, or of each corresponding RNA species, measured in a) to        the average level of each of the low affinity EGFR ligands, or        of each corresponding RNA species measured in b)        where if the average level of low affinity EGFR ligands, or RNAs        coding for them, measured in a) is greater than the average        level of high affinity EGFR ligands, or RNAs coding for them,        measured in b), the patient is predicted to have the favorable        outcome as a result of treatment #1, as well as as a result of        treatment #2, and if the average level of low affinity EGFR        ligands, or RNAs coding for them, measured in a) is less than or        equal to the average level of high affinity EGFR ligands, or        RNAs coding for them, measured in b), the patient is predicted        to have an unfavorable outcome from treatment #1, but is        predicted to have a favorable outcome from treatment #2.

In one embodiment, the at least two low affinity EGFR ligands is atleast three low affinity ligands and the at least two high affinity EGFRligands is three high affinity ligands. In another embodiment, the atleast two low affinity EGFR ligands is four low affinity ligands and theat least two high affinity EGFR ligands is three high affinity ligands.In another embodiment, the at least two high affinity EGFR ligands isthree high affinity ligands. In another embodiment, the at least two lowaffinity EGFR ligands is four low affinity ligands and the at least twohigh affinity EGFR ligands is at least two high affinity ligands.

In one embodiment, a monoclonal antibody preparation comprising only asingle species of monoclonal antibody is provided for use in thetreatment of a patient predicted to have a favorable outcome fromtreatment with either of the monoclonal anti-EGFR antibody preparationor the oligoclonal anti-EGFR antibody preparation and the patient issubsequently treated with the monoclonal preparation. In anotherembodiment, this patient is subsequently treated with the oligoclonalanti-EGFR antibody preparation.

In yet another embodiment, if the patient is predicted to have anunfavorable outcome from treatment with the monoclonal anti-EGFRantibody preparation, the patient is subsequently treated with theoligoclonal anti-EGFR antibody preparation.

The monoclonal anti-EGFR antibody preparation comprises a singlemonoclonal antibody that may be selected from, e.g., cetuximab,zalutumumab, nimotuzumab, matuzumab and panitumumab, all of which blockligand binding to EGFR, and therefore bind to the same or overlappingEGFRepitopes (Bin 1) and are not suitable for use together inoligoclonal antibody preparations. Members of the plurality of anti-EGFRantibody species in an oligoclonal preparation separately and uniquelybind to two different extracellular epitopes of EGFR, may separately anduniquely bind to at least three extracellular epitopes of EGFR (in somecases no more than three). Such a plurality may comprise two or threedifferent species of monoclonal anti-EGFR antibodies, and in someembodiments no more than three different species. Mixtures comprisingmore than one antibody against any one epitope of EGFR are lesspreferred. In certain embodiments the oligoclonal preparations are duos,trios, or fourfold combinations of antibodies as disclosed in theOligoclonal Applications. In other embodiments, the oligoclonalpreparations provided herein comprise one or more of the anti-EGFRantibodies (e.g., of the above duos or trios or other oligoclonalcombinations) that are not Bin 1 antibodies in combination with one ofcetuximab, zalutumumab, nimotuzumab, matuzumab and panitumumab (whichare all Bin 1 antibodies).

In another embodiment, if the patient is predicted to have anunfavorable outcome from treatment with the monoclonal anti-EGFRantibody preparation, the patient is subsequently treated withcombination therapy comprising separate administration of at least twodifferent monoclonal anti-EGFR antibodies. In certain aspects, the atleast two different monoclonal anti-EGFR antibodies are selected fromany of the anti-EGFR antibodies disclosed in the OligoclonalApplications, as well as from cetuximab, zalutumumab, nimotuzumab,matuzumab and panitumumab, provided that one of the anti-EGFR antibodiesis an antibody that inhibits ligand binding to EGFR.

In another embodiment the tumor is a tumor of the skin, central nervoussystem, head, neck, esophagus, stomach, colon, rectum, anus, liver,pancreas, bile duct, gallbladder, lung, breast, ovary, uterus, cervix,vagina, testis, germ cells, prostate, kidney, ureter, urinary bladder,adrenal, pituitary, thyroid, bone, muscle or connective tissue.

In another aspect, a method of treating a tumor in a patient byadministration of a monoclonal anti-EGFR antibody preparation comprisinga single species of monoclonal antibody is provided, the methodcomprising, prior to the administration, determining that the tumor doesnot have a level of high affinity ligands that is equal to or greaterthan the level of low affinity ligands in the tumor and notadministering the monoclonal preparation if the tumor does have a levelof high affinity ligands that is equal to or greater than the level oflow affinity ligands in the tumor.

In another aspect, a method of treating a tumor in a patient byadministration of an oligoclonal anti-EGFR antibody preparationcomprising a plurality of species of monoclonal anti-EGFR antibodies,one against each of at least two extracellular epitopes of EGFR, one ofthe species of monoclonal anti-EGFR antibody inhibiting the binding ofligand to EGFR, is provided, the method comprising, prior to theadministration, determining that the tumor has a level of high affinityligands that is equal to or greater than the level of low affinityligands in the tumor. In one embodiment, the oligoclonal anti-EGFRantibody preparation is MM-151.

Also provided are kits for testing a tumor biopsy sample to determinelevels of both high and low affinity EGFR ligands in the sample, saidkits being comprised by one or more containers comprising;

-   -   a) at least two pairs of high affinity EGFR ligand-specific        polymerase chain reaction (PCR) primers,    -   b) at least two pairs of low affinity EGFR ligand-specific PCR        primers, and    -   c) at least one reverse transcription PCR (RT-PCR) reagent.

In one embodiment the at least two pairs of high affinity EGFRligand-specific polymerase chain reaction primers are specific to atleast two of betacellulin, EGF, HB-EGF or TGFα and each of the at leasttwo pairs of low affinity EGFR ligand-specific polymerase chain reactionprimers are specific to at least two of amphiregulin, epigen, orepiregulin. In another embodiment the at least two pairs of highaffinity EGFR ligand-specific primers consist of all of betacellulin,EGF, HB-EGF and TGFα and the at least two pairs of high affinity EGFRligand-specific primers consist of all of amphiregulin, epigen, andepiregulin. In another embodiment the kit comprises at least onefluorescent reporter molecule suitable for use in a real-time RT-PCRassay. In yet another embodiment the at least one RT-PCR reagent is oneor more of an RNA-dependent DNA polymerase, a DNA-dependent DNApolymerase, a buffer, or a solution comprising at least micromolarconcentrations of each of adenosine triphosphate (ATP), guanosinetriphosphate (GTP), cytidine triphosphate (CTP), and thymidinetriphosphate (TTP). In a further embodiment the one or more containerscomprises either or both of at least one container that has an internaltemperature of below 20° C. and above 0° C., and at least one containerthat has an internal temperature of below 0° C. Preferably the contentsof all, or at least one, of the at least one container has been preparedunder cGMP conditions.

In another aspect, a method is provided for treating a patient having acancerous tumor, the method comprising determining, according to any ofthe methods described above, if the patient is predicted to have thefavorable outcome as a result of treatment #1 and as a result oftreatment #2 or if the patient is predicted to have the favorableoutcome as a result of treatment #2 but not as a result of treatment #1,wherein if the patient is predicted to have a favorable outcome as aresult of treatment #1 and as a result of treatment #2, the patient istreated with treatment #1 or treatment #2; and if the patient ispredicted to have a favorable outcome as a result of treatment #2 butnot as a result of treatment #1, the patient is treated with treatment 2and not with treatment #1. Determining whether the patient will have afavorable outcome may be accomplished by use of any of the theranosticmethods described above, which may be accomplished by use of a kitdescribed above.

In certain aspects of the above methods, the oligoclonal anti-EGFRantibody preparation is a composition comprising a trio of anti-EGFRantibodies comprising a first antibody, a second antibody and a thirdantibody, wherein (i) the first antibody is, or competes for binding toEGFR with, or binds to the same epitope as, an antibody selected fromthe group consisting of ca, cb and cc; (ii) the second antibody is, orcompetes for binding to EGFR with, or binds to the same epitope as, anantibody selected from the group consisting of cd, ce and cf; and (iii)the third antibody is, or competes for binding to EGFR with, or binds tothe same epitope as, an antibody selected from the group consisting ofcg, ch, ci, cj and ck, wherein ca, cb, cd, ce, cf, cg, ch, ci, cj, andck are each disclosed in PCT Int. Pub. No. WO/2011/140254 andcorresponding pending U.S. patent application Ser. No. 13/100,920.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C: Phospho-EGF receptor and phospho-ERK signaling inhibitionby single and pairwise combinations of Bin 1+Bin 2 or Bin 1+Bin 3antibodies and comparisons with other known anti-EGFR antibodies such ascetuximab, nimotuzumab, and zalutumumab. FIG. 1A shows inhibition of ERKactivation by the Bin1/2 antibodies cb and cd. FIG. 1B shows theinhibition of EGFR activation by the Bin1/2 antibodies and cd. FIG. 1Cshows the inhibition of ERK activation by the Bin1/3 antibodies cb andch. Lines depict a five parameter logistic fit to the data from eachcombination.

FIGS. 2A-2G: Inhibition of ligand-mediated tumor cell signaling in A431cells preincubated with varying concentrations of anti-EGFR monoclonalantibodies cb (Bin1), cd (Bin2), cetuximab, zalutumumab, or nimotuzumab;as well as the oligoclonal combination of cb+cd; for 2 hrs. Afterincubation cells were stimulated with an EGFR ligand (8 nanomolar finalconcentration) for about 10 minutes. Figures show ELISA analysis ofphospho-ERK (pERK) production (y-axis) as a function of antibodyconcentration (x-axis, in Log Molar concentration) after stimulationwith the ligands amphiregulin (FIG. 2A), epigen (FIG. 2B), epiregulin(FIG. 2C), betacellulin (FIG. 2D), epidermal growth factor (EGF, FIG.2E), heparin-binding EGF-like growth factor (HB-EGF, FIG. 2F), ortransforming growth factor α (TGF-α, FIG. 2G). A431 cells incubated inthe absence of anti-EGFR antibodies but with the ligand indicated ineach graph (+Lig) or without ligand stimulation (−Lig) were used aspositive and negative controls, respectively.

FIGS. 3A-3G: Inhibition of ligand-mediated tumor cell signaling in A431cells preincubated with varying concentrations of anti-EGFR monoclonalantibodies cb (Bin1), ch (Bin3), cetuximab, zalutumumab, or nimotuzumab;as well as the oligoclonal combination of cb+ch; for 2 hrs. Afterpre-incubation with antibodies, cells were stimulated with an EGFRligand (8 nanomolar final concentration) for 10 minutes. Figures showELISA analysis of phospho-ERK (pERK) production (y-axis) as a functionof antibody concentration (x-axis, in Log Molar concentration) afterstimulation with the ligands amphiregulin (FIG. 3A), epigen (FIG. 3B),epiregulin (FIG. 3C), betacellulin (FIG. 3D), epidermal growth factor(EGF, FIG. 3E), heparin-binding EGF-like growth factor (HB-EGF, FIG.3F), or transforming growth factor α (TGF-α, FIG. 3G). A431 cellsincubated in the absence of anti-EGFR antibodies but with the ligandindicated in each graph (+Lig) or without ligand stimulation (−Lig) wereused as positive and negative controls, respectively.

FIGS. 4A-4L: Inhibition of high affinity EGFR ligand-mediated tumor cellproliferation. H322M cells (FIGS. 4A-4D), H1975 cells (FIGS. 4E-4H), andLIM1215 cells (FIGS. 4I-4L) were treated with varying concentrations ofanti-EGFR monoclonal and oligoclonal antibodies in the presence of EGFRligands. Cells were treated with 200 ng/ml amphiregulin (AREG) (FIGS.4A, 4E, and 4I), 50 ng/ml EGF (FIGS. 4B, 4F, and 4J), 50 ng/ml TGFα(FIGS. 4C, 4G, and 4K) or 90 ng/ml HB-EGF (FIGS. 4D, 4H, and 4L) in thepresence of varying concentrations of MM-151 (open circles or cetuximab(CTX, solid squares; Bristol-Myers Squibb). Cells treated with ligand(+Lig, upward arrow) or without ligand (−Lig, downward arrow) in theabsence of antibody treatment served as controls. The y-axes representcell viability as the fraction of the viability of theamphiregulin-treated control cells and the x-axes represent antibodyconcentration in Log (Molar).

FIGS. 5A-5L: Effect of EGFR high affinity ligand titration on cellresponsiveness to anti-EGFR inhibitors in vitro. The non-small cell lungcancer (NSCLC) lines H322M (FIGS. 5A-5D), HCC827 (FIGS. 5E-5H), andH1975 (FIGS. 5I-5L) were tested. Controls were growth in media withamphiregulin alone (+AREG, 200 ng/ml) or EGF alone as a control (+EGF,20 ng/ml) or no added ligand (−Lig). Treatments were with varyingconcentrations (0.1-1 μM final concentration) of MM-151 or cetuximab(CTX) in the following conditions: amphiregulin alone (200 ng/ml, FIGS.5A, 5E, and 5I); a 1000:1 amphiregulin:EGF ratio (0.2 ng/ml EGF, FIGS.5B, 5F, and 5J); a 100:1 amphiregulin:EGF ratio (2 ng/ml EGF, FIGS. 5C,5G, and 5K); and a 10:1 amphiregulin:EGF ratio (20 ng/ml EGF, FIGS. 5D,5H, and 5L). The y-axes represent cell viability as a fraction of theviability of the AREG-treated control cells, whereas and the x-axesrepresent antibody concentration in Log (Molar).

FIG. 6: Effect of EGFR ligand concentration on phopho-ERK cellsignaling. The epidermoid cancer cell line A431 was treated with mediaalone (“No Inhibitor”), MM-151 (100 nM) or cetuximab (100 nM) for 2 hrs,followed by the addition of various concentrations of EGF (0.16 ng/ml,0.8 ng/ml, 4.0 ng/ml, 20 ng/ml, 100 ng/ml) or AREG (0.48 ng/ml, 2.4ng/ml, 12 ng/ml, 60 ng/ml, 300 ng/ml), alone or in combination. Cellswere incubated with the various EGF and AREG ligand combinations for 10minutes, lysed, and levels of ERK phosphorylation measured byphospho-ERK ELISA.

DETAILED DESCRIPTION I. Definitions

The terms “EGFR,” and “EGF receptor” are used interchangeably herein torefer to human EGFR protein (also referred to as ErbB1 or HER1); seeUniProtKB/Swiss-Prot entry P00533.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Such antibodies may be obtained, e.g. from hybridomas orby recombinant expression. Antigen binding fragments (including scFvs)of such immunoglobulins are also encompassed by the term “monoclonalantibody” as used herein. Monoclonal antibodies are highly specific,generally being directed against a single epitope on a single antigensite, e.g., on the extracellular domain of EGFR. Monoclonal antibodiesinclude chimeric antibodies - whose variable regions derive from a firstanimal species (e.g., mouse) and whose constant regions derive from asecond animal species (e.g., human), human antibodies and humanizedantibodies.

The terms “treat,” “treating,” and “treatment,” as used herein, refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration to a subject, an antibody or antibodypair or trio disclosed herein, for example, a subject having a disorderassociated with EGFR dependent signaling or predisposed to having such adisease or disorder, in order to prevent, cure, delay, reduce theseverity of, or ameliorate one or more symptoms of the disease ordisorder or recurring disease or disorder, or in order to prolong thesurvival of a subject beyond that expected in the absence of suchtreatment.

Commercially available pharmaceutical anti-EGFR antibodies includecetuximab, panitumumab and nimotuzumab (which is not yet available inthe US market). Other pharmaceutical anti-EGFR antibodies includezalutumumab, and matuzumab, which are in development. Still otheranti-EGFR antibodies include those disclosed in the OligoclonalApplications, e.g., the antibodies disclosed below.

P1X is a human IgG1 having a heavy chain variable region comprising SEQID NO: 1 and a light chain variable region comprising SEQ ID NO: 2;

P2X is a human IgG1 having a heavy chain variable region comprising SEQID NO: 3 and a light chain variable region comprising SEQ ID NO: 4; and

P3X is a human IgG1 having a heavy chain variable region comprising SEQID NO: 5 and a light chain variable region comprising SEQ ID NO: 6.

“MM-151” indicates a triple combination of P1X+P2X+P3X at a P1X:P2X:P3Xmolar ratio of 2:2:1.

TABLE 1 Exemplary Antibodies P1X V_(H) MGFGLSWLFLVAILKGVQC SEQ ID NO: 1QVQLVQSGAEVKKPGSSVKV SCKASGGTFSSYAISWVRQA PGQGLEWMGSIIPIFGTVNYAQKFQGRVTITADESTSTAY MELSSLRSEDTAVYYCARDP SVNLYWYFDLWGRGTLVTVSSP1X V_(L) MGTPAQLLFLLLLWLPDTTG SEQ ID NO: 2 DIQMTQSPSTLSASVGDRVTITCRASQSISSWWAWYQQKP GKAPKLLIYDASSLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQYHAHPTTFGG GTKVEIK P2X V_(H) MGFGLSWLFLVAILKGVQC SEQ ID NO: 3QVQLVQSGAEVKKPGSSVKV SCKASGGTFGSYAISWVRQA PGQGLEWMGSIIPIFGAANPAQKSQGRVTITADESTSTAY MELSSLRSEDTAVYYCAKMG RGKVAFDIWGQGTMVTVSS P2X V_(L)MGTPAQLLFLLLLWLPDTTG SEQ ID NO: 4 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSPNNKNYLA WYQQKPGQPPKLLIYWASTR ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYGS PITFGGGTKVEIK P3X V_(H) MGFGLSWLFLVAILKGVQCSEQ ID NO: 5 QVQLVQSGAEVKKPGASVKV SCKASGYAPTSYGINWVRQAPGQGLEWMGWISAYNGNTYY AQKLRGRVTMTTDTSTSTAY MELRSLRSDDTAVYYCARDLGGYGSGSVPFDPWGQGTLVTVSS P3X V_(L) MGTPAQLLFLLLLWLPDTTG SEQ ID NO: 6EIVMTQSPATLSVSPGERAT LSCRASQSVSSNLAWYQQKP GQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQS EDFAVYYCQDYRTWPRRVFG GGTKVEIK zalutumumab QVQLVESGGGVVQPGRSLRLS SEQ ID NO: 7 V_(H) CAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDS VKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSS zalutumumab  AIQLTQSPSSLSASVGDRVTI SEQ ID NO: 8V_(L) TCRASQDISSALVWYQQKPGK APKLLIYDASSLESGVPSRFS GSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVE IK Nimotuzumab  QVQLQQPGAELVKPGASVKLS SEQ ID NO: 9V_(H) CKASGYTFTNYYIYWVKQRPG QGLEWIGAGINPTSGGSNFNE KFKTKATLTVDESSTTAYMQLSSLTSEDSAVYYCTRQGLWFD SDGRGFDFWGQGTTLTVSS Nimotuzumab DVLMTQIPLSLPVSLGDQASISC SEQ ID NO: 10 V_(L) RSSQNIVHSNGNTYLDWYLQKPGQSPNLLIYKVSNRESGVPDRF RGSGSGTDFTLKISRVEAEDLGV YYCFQYSHVPWTFGGGTKLEIK

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

The term “sample” refers to tissue, body fluid, or a cell (or a fractionof any of the foregoing) taken from a patient. Normally, the tissue orcell will be removed from the patient, but in vivo diagnosis is alsocontemplated. In the case of a solid tumor, a tissue sample can be takenfrom a surgically removed tumor and prepared for testing by conventionaltechniques. In the case of lymphomas and leukemias, lymphocytes,leukemic cells, or lymph tissues can be obtained (e.g., leukemic cellsfrom blood) and appropriately prepared. Other samples, including urine,tears, serum, plasma, cerebrospinal fluid, feces, sputum, cell extractsetc. can also be useful for particular cancers.

Various aspects of the disclosure are described in further detail in thefollowing subsections.

II. Outcomes

A patient having a tumor predicted by the methods disclosed herein tohave a favorable outcome following treatment with a monoclonal oroligoclonal anti-EGFR antibody, and who is then treated accordingly, mayexhibit one of the following responses to therapy:

-   Pathologic complete response (pCR): absence of invasive cancer    following primary systemic treatment.-   Complete Response (CR): Disappearance of all target lesions.-   Partial Response (PR): At least a 30% decrease in the sum of    dimensions of target lesions, taking as reference the baseline sum    diameters; or-   Stable Disease (SD): Neither sufficient shrinkage to qualify for    partial response, nor sufficient increase to qualify for progressive    disease, taking as reference the smallest sum diameters while on    study.

In exemplary outcomes, patients treated as disclosed herein mayexperience improvement in at least one sign of cancer.

In one embodiment the patient so treated exhibits pCR, CR, PR, or SD.

In another embodiment, the patient so treated experiences tumorshrinkage and/or decrease in growth rate, i.e., suppression of tumorgrowth. In another embodiment, unwanted cell proliferation is reduced orinhibited. In yet another embodiment, one or more of the following canoccur: the number of cancer cells can be reduced; tumor size can bereduced; cancer cell infiltration into peripheral organs can beinhibited, retarded, slowed, or stopped; tumor metastasis can be slowedor inhibited; tumor growth can be inhibited; recurrence of tumor can beprevented or delayed; one or more of the symptoms associated with cancercan be relieved to some extent.

In other embodiments, such improvement is measured by a reduction in thequantity and/or size of measurable tumor lesions. Measurable lesions aredefined as those that can be accurately measured in at least onedimension (longest diameter is to be recorded) as ≧10 mm by CT or MRIscan (e.g., CT scan slice thickness no greater than 5 mm), 10 mm calipermeasurement by clinical exam or >20 mm by chest X-ray. The size ofnon-target lesions can also be measured for improvement. In oneembodiment, lesions can be measured on x-rays or CT or MRI images.

In other embodiments, cytology or histology can be used to evaluateresponsiveness to a therapy. The cytological confirmation of theneoplastic origin of any effusion that appears or worsens duringtreatment when the measurable tumor has met criteria for response orstable disease can be considered to differentiate between response orstable disease (an effusion may be a side effect of the treatment) andprogressive disease.

In some embodiments, a beneficial response to therapy is indicated by atleast one therapeutic effect selected from the group consisting ofreduction in size of a tumor, reduction in number of metastatic lesionsappearing over time, complete remission, partial remission, stabledisease, increase in overall response rate, or a pathologic completeresponse.

III. Pharmaceutical Compositions

Pharmaceutical compositions for use in the methods provided for hereinare commercially available anti-EGFR compositions, e.g., of cetuximab,panitumumab and nimotuzumab, as well as the various pharmaceuticalcompositions provided in the Oligoclonal Applications.

IV. Use of Oligoclonal Antibodies

Provided herein are methods of determining whether or not a monoclonalanti-EGFR antibody preparation comprising only a single species ofanti-EGFR antibody should be used to treat a tumor. Use of oligoclonalanti-EGFR antibodies for the treatment of a disease associated withhigh-affinity EGFR ligand-driven signaling is also provided, as aremethods of use of oligoclonal anti-EGFR antibodies for the treatment oftumor comprising protein or mRNA levels of at least two high-affinityEGFR ligands that are higher than levels in the tumor of at least twolow-affinity EGFR ligands. Cancers treated in accordance with themethods provided include melanoma, breast cancer, ovarian cancer, renalcarcinoma, gastrointestinal cancer, gastro-esophageal junction cancer,colon cancer, lung cancer, pancreatic cancer, skin cancer, head and neckcancer glioblastoma, prostate cancer and other solid and/or metastatictumors.

The monoclonal or oligoclonal antibody can be administered alone or withanother therapeutic agent that acts in conjunction with orsynergistically with the oligoclonal antibody to treat the diseaseassociated with EGFR-mediated signaling.

Also provided are kits for testing a tumor sample, e.g., a tumor biopsysample or a circulating tumor cell, to determine levels of both high andlow affinity EGFR ligands in the sample, said kits being comprised byone or more containers comprising;

-   -   a) at least two pairs of high affinity EGFR ligand-specific        polymerase chain reaction (PCR) primers,    -   b) at least two pairs of low affinity EGFR ligand-specific PCR        primers, and    -   c) at least one reverse transcription PCR (RT-PCR) reagent.

In another embodiment, the kit may further contain instructions for usein determining how to treat a tumor in a patient following determinationof levels of high and low affinity ligands in a sample of the tumor. Thekit may include an indication of the intended use of the contents of thekit (e.g., in the form of a label or other printed or recorded matter).

Other embodiments are described in the following non-limiting Examples.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents ofSequence Listing, figures and all references, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

EXAMPLES Materials and Methods

Throughout the examples, the following materials and methods are usedunless otherwise stated.

In general, unless otherwise indicated, conventional techniques ofchemistry, molecular biology, recombinant DNA technology, immunology(especially, e.g., antibody technology), and standard techniques inpolypeptide preparation are used. See, e.g., Sambrook, Fritsch andManiatis, Molecular Cloning Cold Spring Harbor Laboratory Press (1989);Antibody Engineering Protocols (Methods in Molecular Biology), 510,Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach(Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996);Antibodies: A Laboratory Manual, Harlow et al., C.S.H.L. Press, Pub.(1999); and Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons (1992).

Pulverization of Tumor Cells

A cryopulverizer (COVARIS Inc.) is used for the pulverization of tumors.Tumors are stored in special bags (pre-weighed before the addition ofthe tumor) and placed in liquid nitrogen while handling them. For smalltumors, 200 μL of Lysis buffer is first added to the bag containing thetumor, frozen in liquid nitrogen and then pulverized to improve therecovery of the tumor from the bag. Pulverized tumors are transferred to2 mL EPPENDORF tubes and placed in liquid nitrogen until ready forfurther processing.

Lysis of Tumor Cells

Tumors are lysed in Lysis buffer supplemented with protease andphosphatase inhibitors. Lysis Buffer is added to the tumor aliquots in afinal concentration of about 62.5 mg/mL. Tumor samples are homogenizedby vortexing for 30 sec and incubating on ice for about 30 min. Thelysates are spun for about 10 min in Qiagen QIASHREDDER columns forfurther homogenization of the samples. Cleared lysates are aliquotedinto fresh tubes for further processing.

Measurement of Inhibition of EGFR Ligand-Mediated Phosphorylation of ERKin Tumor Cells

Inhibition of ligand-mediated tumor cell signaling is investigated asfollows: A431 (ATCC CRL-1555™) epidermoid carcinoma cells are seeded ata density of 35,000 cells/well or 17,500 cells per half well in 96 welltissue culture plates and grown in DMEM medium supplemented withantibiotics, 2 mM L-glutamine and 10% fetal bovine serum (FBS) for 24hours at 37° C. and 5% carbon dioxide. Cells are serum starved in 1% FBSmedium with antibiotics and 2 mM L-glutamine for about 20 hours at 37°C. and 5% carbon dioxide. Cells are then treated as described below ineach Example. Cells are washed with ice-cold PBS and lysed in 50 μlice-cold Lysis buffer (Mammalian Protein Extraction Lysis Reagent(M-PER, Pierce, Thermo Scientific product #78505) amended with 150 mMNaCl and protease inhibitor cocktail (Sigma, P714)) by incubating on icefor 30 minutes. Lysates are either analyzed immediately by ELISA forphospho-ERK (a downstream effector of EGFR) or frozen at '180° C. untiluse.

ELISA Assays

For the phospho-EGFR sandwich ELISA, 96-half well GREINER high bindingplates (Cat. #675077; GREINER BIO-ONE, Monroe, N.C.) are coated with 50μL of an EGFR antibody (4 μg/ml final concentration; EGFR Ab-11, Clone:199.12, without BSA and azide, Fisher Scientific, cat #MS396P1ABX), andincubated overnight at room temperature. Next morning, plates are washed3 times with 100 μl/well PBST (0.05% Tween-20) on a BIOTEK plate washer.Plates are subsequently blocked for about 1 hour at room temperaturewith 2% BSA in PBS. The plates are washed 3 times with 100 μl/well PBST(0.05%Tween-20) on the BIOTEK plate washer. Cell lysates (50 μl) orstandards (pEGFR pY1068 ELISA kit, R&D Systems, cat #DYC3570) diluted in50% Lysis buffer and 1%BSA-PBS (per the manufacturer's recommendations)are added to the plates in duplicates and incubated for 2 hrs at roomtemperature or overnight at 4° C. with shaking. Plates are then washed 3times with 100 μl/well in the BIOTEK plate washer with PBST (PBS with0.05%Tween-20). About 50 μl of a detection antibody (pEGFR pY1068 ELISAkit, R&D Systems, cat #DYC3570) conjugated to horseradish peroxidase(HRP) diluted (as per manufacturer's instructions) in 2% BSA, PBS isadded and incubated for about 2 hour at room temperature. The plate iswashed 3 times with 100 μl/well in the BIOTEK plate washer with PBST(0.05%Tween-20). About 50 μL of SUPERSIGNAL PICO ELISA substrate isadded and the plate is read using an Envision (Perkin Elmer) platereader. The data are analyzed and duplicate samples are averaged anderror bars are used to represent the standard deviation between the tworeplicates.

The phospho-ERK ELISA is performed similarly to the phospho-EGFR ELISAwith the following changes: Human pERK ELISA DUOSET kit is purchasedfrom R&D Systems (cat #DYC1018-5) and used as recommended by themanufacturer. The data are analyzed by subtracting background signal,regressing to a recombinant standard supplied by the manufacturer, andback-calculating the data (BCD) to correct for dilution factors.Duplicate samples are averaged and error bars are used when indicated torepresent the standard deviation between two replicates.

Example 1 Phospho-EGF Receptor and Phospho-ERK Signaling Inhibition bySingle and Pairwise Combinations of Bin 1+Bin 2 or Bin 1+Bin 3Antibodies and Comparisons with Each of Individual Monoclonal AntibodiesCetuximab, Nimotuzumab, and Zalutumumab

A431 cells were treated with single antibodies or antibody pairs andtheir ability to inhibit EGFR-dependent signaling was compared to thateach of cetuximab, nimotuzumab, and zalutumumab. Cells were incubatedwith varying concentrations of anti-EGFR antibodies for 2 hrs, and thenstimulated with an EGFR ligand for 10 minutes at 37° C. and 5% carbondioxide. The seven recombinant human EGFR ligands used individually were100 ng/ml amphiregulin (“AREG,” R&D Systems, cat #262-AR/CF), 100 ng/mlbetacellulin (R&D Systems, cat #261-CE-050/CF), EGF (PreproTech, cat#AF-100-15), 220 ng/ml epigen (epithelial mitogen homolog, PreproTech,cat #100-51), 150 ng/ml epiregulin (R&D Systems, cat #1195-EP/CF), 90ng/ml HB-EGF (heparin-binding EGF-like growth factor, PreproTech, cat#100-47), and 50 ng/ml TGFα (transforming growth factor alpha, R&DSystems, cat #239-A). ELISA measurements were performed as describedabove for pERK and pEGFR signaling and the results are shown in FIGS.1A-C. Only mixtures of Bin1/Bin2 antibodies cb and cd (FIG. 1A) andBin1/Bin3 antibodies cb and ch (FIG. 1C) were effective at completelyinhibiting phospho-ERK signaling when compared to cetuximab,nimotuzumab, and zalutumumab, as well as to individual components cb,cd, and ch. All antibodies, including the mixtures, were effective atcomplete inhibition of Phospho-EGF receptor signaling, with theexception of nimotuzumab (FIG. 1B).

Example 2 Phospho-ERK Signaling Inhibition by Single and PairwiseCombinations of Bin 1, Bin 2, and Bin 3 Antibodies and Comparisons withCetuximab, Nimotuzumab, and Zalutumumab

Single antibodies cb, cd, and ch, or pairs of cb and cd or cb and ch,(as described above in Example 1) were used to treat A431 cells atindicated total concentrations, and their ability to inhibit EGFRligand-dependent signaling was compared to that of each single anti-EGFRantibodies cetuximab, nimotuzumab, and zalutumumab at the sameconcentrations. Cells were incubated with antibody for 2 hours followedby stimulation with EGFR ligand for 10 minutes. Seven EGFR ligands wereused individually: amphiregulin (100 ng/ml), betacellulin (100 ng/ml),EGF (50 ng/ml), epigen (220 ng/ml), epiregulin (150 ng/ml), HB-EGF (90ng/ml), and TGFα (50 ng/ml). Experiments were performed as describedabove and the results are shown in FIGS. 2A-G and 3A-G. Individually, cband cd, as well as well cetuximab, nimotuzumab, and zalutumumab, wereeffective at inhibiting phospho-ERK signaling (i.e., inhibitingphosphorylation of ERK1 and ERK2) in response to the three ligands withlow affinity for EGF receptor (amphiregulin, epigen, and epiregulin),but not in response to the four ligands with high affinity for EGFreceptor (betacellulin, EGF, HB-EGF, and TGFα). Only oligoclonalmixtures of Bin1/Bin2 antibodies cb and cd (FIGS. 2A-G) and Bin1/Bin3antibodies cb and ch (FIGS. 3A-G) were effective at essentiallycompletely inhibiting phospho-ERK signaling in response to all seven(both high- and low-affinity) EGFR ligands when compared to individualcomponents of the mixtures, cb, cd, and ch and the other testedindividual monoclonal antibodies, cetuximab, nimotuzumab, andzalutumumab.

Example 3 Effect of EGFR Ligand Concentration on Phospho-ERK CellSignaling

Inhibition of tumor cell signaling in vitro is analyzed by the methodsdescribed above or minor variations thereof. The epidermoid cancer cellline A431 was treated with media alone (“No Inhibitor”), MM-151 (100 nM)or cetuximab (100 nM) for 2 hrs, followed by the addition of variousconcentrations of EGF (0.16 ng/ml, 0.8 ng/ml, 4.0 ng/ml, 20 ng/ml, 100ng/ml) or AREG (0.48 ng/ml, 2.4 ng/ml, 12 ng/ml, 60 ng/ml, 300 ng/ml),alone or in combination, as shown in FIG. 6. Cells were incubated withthe various EGF and AREG ligand combinations for 10 minutes, lysed, andlevels of ERK phosphorylation measured by phospho-ERK ELISA and analyzedas indicated in the methods. FIG. 6 shows MM-151 and cetuximab-mediatedmodulation of ERK signaling represented as fraction of the highestsignal across all treatments. Phospho-ERK signaling is inhibited bycetuximab in A431 cells under low-affinity EGFR ligand (AREG)stimulation, but become increasingly resistant to inhibition upon theaddition of increasing amounts of the high-affinity EGFR ligand, EGF(middle panel), while inhibition of signaling by MM-151 is largelymaintained under all conditions (lower panel).

Example 4 Inhibition of Tumor Cell Proliferation in the Presence of Highor Low Affinity EGFR Ligands Inhibition of Tumor Cell Proliferation InVitro

Inhibition of cellular proliferation of cells expressing EGFR isexamined in vitro as follows: H322M (NCI, Frederick, Md. 21701), H1975(ATCC CRL-2868™), and LIM1215 (Cell Bank Australia, NSW 2145) cancercells are separately seeded in 96 well tissue culture plates at 5,000cells per well and grown in RPMI-1640 medium supplemented withantibiotics, 2 mM L-glutamine and 10% fetal calf serum (FCS) (H322M andH1975) or RPMI-1640 medium supplemented with 25 mM HEPES, antibiotics, 2mM L-glutamine, 10% FCS, 0.6 μg/ml insulin, 1 μg/ml hydrocortisone and10 μM thioglycerol (LIM1215) for 24 hours at 37 degrees Celsius and 5%carbon dioxide. Medium is then switched to RPMI-1640 with antibiotics, 2mM L-glutamine, 1% FBS (for H322M and H1975) or RPMI-1640 with 25 mMHEPES, antibiotics, 2 mM L-glutamine, 1% FCS, 0.6 μg/ml Insulin, 1 μg/mlhydrocortisone and 10 μM thioglycerol (for LIM1215) supplemented with200 ng/ml AREG, 50 ng/ml EGF, 50 ng/ml TGFα or 90 ng/ml HB-EGF in thepresence of varying concentrations of MM-151 or cetuximab (Bristol-MyersSquibb). Cell viability is measured 72 hours post-treatment using theCellTiter-Glo® (CTG) Luminescent Viable Cell Number Assay (PromegaCorporation) according to manufacturer's instructions. The CTG assaymeasures the number of viable cells in culture based upon quantitationof ATP present, which is an indicator of metabolically active cells.Control treatments include cells treated with 1% FCS-containing medium(as detailed above) in the presence (“+Lig”) or absence (“−Lig”) of therespective ligand treatment. Viable cell numbers are plotted in GraphPadPrism (GraphPad Software, La Jolla, Calif.) as a fraction of therespective ligand (“+Lig”) treatment control.

Results

The non-small cell lung cancer (NSCLC) lines H322M and H1975 and coloncancer cell line LIM1215 were treated with varying concentrations ofMM-151or cetuximab (0.1-1 μM final concentration). Potent inhibition ofgrowth of H322M, H1975 and LIM1215 cells was obtained over a range ofMM-151 concentrations in the presence of high affinity EGFR ligands(EGF, TGFα, HB-EGF), but not in the presence of cetuximab or in assaymedium alone (1% FCS)—FIG. 4 (B-D, F-H, and J-L). Potent inhibition ofgrowth of H322M, H1975 and LIM1215 cells was obtained over a range ofconcentrations for both MM-151 and cetuximab, but not by assay mediumalone (1% FCS) in the presence of the low affinity EGFR ligandamphiregulin (AREG)—FIG. 4 (A, E and I). These data demonstrate theability of the MM-151 oligoclonal mixture to inhibit tumor cellproliferation in vitro in response to both high-affinity (EGF, TGFα,HB-EGF) and low-affinity (AREG) ligands, whereas cetuximab is onlypotently effective in cells treated with low-affinity (AREG) ligand.

Example 5 Effects of EGF Ligand Concentration on Cell Proliferation

Using methods essentially as described in the preceding Example,non-small cell lung cancer (NSCLC) cell lines H322M, HCC827 and H1975were treated with AREG alone (200 ng/ml) or with AREG plus increasingamounts of EGF (0.2, 2, 20 ng/ml) in the presence of varyingconcentrations of MM-151or cetuximab (0.1-1 μM final concentration).

Results

The NSCLC cell lines respond to cetuximab under low-affinity EGFR ligandstimulation (AREG), but become increasingly unresponsive to treatmentupon the addition of increasing amounts of the high-affinity EGFR ligandEGF, while sensitivity to MM-151 is largely maintained (see FIGS.5A-5L).

Example 6 Assays and Kits Measurement of EGFR Family Ligand ExpressionLevels by RT-qPCR

Measurement of EGFR ligand expression in tumor biopsy samples byreal-time quantitative polymerase chain reaction (RT-qPCR) of DNAsreverse transcribed from RNAs is carried out as follows:

Total RNA is isolated from patient biopsy/tumor samples, e.g., bycommercially available standard methods. The method of total RNAisolation may be any method (including conventional methods) suitablefor use with the type of patient biopsy sample being tested, e.g.,fresh, fixed, frozen, formalin fixed paraffin embedded (FFPE), etc.Total RNA is then converted to cDNA using the gene specific primersdescribed below and Qiagen® OneStep RT-PCR reagents and protocol (Cat.#210210, Qiagen, Germantown, Md.). The cDNA is then used for RT-qPCRusing the following gene specific primers as TaqMan® probe sets obtainedfrom Applied Biosystems (Carlsbad, Calif.) along with reagents andequipment from the same source, all as described below:

-   -   1. TaqMan® Gene Expression Assay, Gene Name: betacellulin, Assay        ID: Hs01101201_ml    -   2. TaqMan® Gene Expression Assay, Gene Name: transforming growth        factor, alpha, Assay ID: Hs00608187_ml    -   3. TaqMan® Gene Expression Assay, Gene Name: heparin-binding        EGF-like growth factor, Assay ID: Hs00181813_ml    -   4. TaqMan® Gene Expression Assay, Gene Name: epiregulin, Assay        ID: Hs00914313_ml    -   5. TaqMan® Gene Expression Assay, Gene Name: amphiregulin, Assay        ID: Hs00950669_ml    -   6. TaqMan® Gene Expression Assay, Gene Name: epidermal growth        factor, Assay ID: Hs01099999_ml.    -   7. TaqMan® Gene Expression Assay, Gene Name: epithelial mitogen        homolog (epigen), Assay ID Hs02385425_ml.        5 μl of diluted cDNA is mixed with 10 μl of TaqMan® Fast        Advanced Master Mix (Cat. #4444556), 2 μl of the above primer        probe set and 3 μl of water in a MicroAmp® Fast Optical 96-Well        Reaction Plate (Cat. #4366932). The plate is then placed in a        Viia™ 7 RT-qPCR machine and a thermal cycling program completed        as described in the manufacturers protocol. Data collection and        analysis is carried out using the Viia™ 7-RUO-Software (Applied        Biosystems).

Also see US Patent Publication Nos. 20030165952, 20040009489,20050095634, 20050266420, 20070141587, 20070141588, 20070141589,20080182255, 20090125247, 20090280490, 20100221754 and 20110086349, andU.S. Pat. Nos. 6,750,013, 6,808,888, 6,939,670, 6,964,850, 6,692,916,7,081,340, 7,171,311, 7,526,387, 7,569,345, 7,622,251, 7,871,769,7,838,224, 7,858,304, 7,930,104, and 8,071,286.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments described herein. Such equivalents are intended to beencompassed by the following claims. Any combination of the embodimentsdisclosed in the any plurality of the dependent claims is contemplatedto be within the scope of the disclosure.

What is claimed is:
 1. A kit for testing a tumor sample to determinelevels of both high and low affinity EGFR ligands in the sample, saidkit comprising a container comprising; a) at least two pairs of highaffinity EGFR ligand-specific polymerase chain reaction (PCR) primers,b) at least two pairs of low affinity EGFR ligand-specific PCR primers,and c) at least one reverse transcription PCR (RT-PCR) reagent.
 2. Thekit of claim 1, wherein the at least two pairs of high affinity EGFRligand-specific polymerase chain reaction primers are specific to atleast two of betacellulin, EGF, HB-EGF or TGF alpha and each of the atleast two pairs of low affinity EGFR ligand-specific polymerase chainreaction primers are specific to at least two of amphiregulin, epigen,or epiregulin.
 3. The kit of claim 2, wherein the at least two pairs ofhigh affinity EGFR ligand-specific primers comprise specific primerpairs for betacellulin, EGF, HB-EGF and TGF alpha and the at least twopairs of high affinity EGFR ligand-specific primers comprise specificprimer pairs for all of amphiregulin, epigen, and epiregulin.
 4. The kitof claim 2, further comprising at least one fluorescent reportermolecule.
 5. The kit of claim 1, wherein the at least one RT-PCR reagentis one or more of an RNA-dependent DNA polymerase, a DNA-dependent DNApolymerase, or a solution comprising at least a micromolar concentrationof each of ATP, GTP, CTP and TTP.
 6. The kit of claim 2, wherein the atleast one RT-PCR reagent is one or more of an RNA-dependent DNApolymerase, a DNA-dependent DNA polymerase, or a solution comprising atleast a micromolar concentration of each of ATP, GTP, CTP and TTP. 7.The kit of claim 5 wherein the primers, the reagents, or both, have beenprepared under current Good Manufacturing Practice (cGMP) conditions. 8.The kit of claim 5, wherein container further comprises total RNAisolated from a tumor sample.
 9. The kit of claim 8, wherein the tumorsample from which the RNA is isolated is a fresh, fixed, frozen, orformalin fixed paraffin embedded (FFPE) sample.
 10. The kit of claim 9,wherein the tumor sample is a circulating tumor cell sample.
 11. The kitof claim 9, wherein the tumor sample is a biopsy sample.
 12. The kit ofclaim 9, wherein the tumor sample is from a tumor of the skin, centralnervous system, head, neck, esophagus, stomach, colon, rectum, anus,liver, pancreas, bile duct, gallbladder, lung, breast, ovary, uterus,cervix, vagina, testis, germ cells, prostate, kidney, ureter, urinarybladder, adrenal, pituitary, thyroid, bone, muscle or connective tissue.13. A kit for testing a tumor sample to determine levels of both highand low affinity EGFR ligands in the sample, said kit comprising one ormore containers, one of said containers comprising; a) at least twopairs of high affinity EGFR ligand-specific polymerase chain reaction(PCR) primers, b) at least two pairs of low affinity EGFRligand-specific PCR primers, c) at least one reverse transcription PCR(RT-PCR) reagent, and d) RNA isolated from a tumor sample.
 14. The kitof claim 13, wherein the container comprising a), b), c), and d) is a 96well plate.
 15. The kit of claim 13, wherein the tumor sample from whichthe RNA is isolated is a fresh, fixed, frozen, or formalin fixedparaffin embedded (FFPE) sample.
 16. The kit of claim 15, wherein thetumor sample is a circulating tumor cell sample.
 17. The kit of claim16, wherein the tumor sample is a biopsy sample.
 18. The kit of claim15, wherein the tumor sample was from a tumor of the skin, centralnervous system, head, neck, esophagus, stomach, colon, rectum, anus,liver, pancreas, bile duct, gallbladder, lung, breast, ovary, uterus,cervix, vagina, testis, germ cells, prostate, kidney, ureter, urinarybladder, adrenal, pituitary, thyroid, bone, muscle or connective tissue.19. The kit of claim 15, wherein the container comprising a), b), c),and d) is a 96 well plate
 20. The kit of claim 18, wherein the containercomprising a), b), c), and d) is a 96 well plate.
 21. A monoclonalanti-EGFR antibody preparation comprising a single species of monoclonalantibody for treatment of a patient predicted by a method to have afavorable outcome from treatment with the monoclonal anti-EGFR antibodypreparation by determining whether a patient having a tumor is predictedto have a favorable outcome, comprising reduction of growth of thetumor, as a result of treatment #1 with a monoclonal anti-EGFR antibodypreparation comprising a single species of monoclonal antibody, and as aresult of treatment #2 with an oligoclonal anti-EGFR antibodypreparation comprising a plurality of species of monoclonal anti-EGFRantibodies, one against each of at least two extracellular epitopes ofEGFR, or, whether the patient is predicted to have the favorable outcomeas a result of treatment #2 but not as a result of treatment #1; themethod comprising: obtaining a biopsy sample of the tumor and: a)measuring levels of protein or mRNAs coding for at least two lowaffinity EGFR ligands selected from amphiregulin, epigen, or epiregulinin the biopsy sample, b) measuring levels of protein or mRNAs coding forat least two high affinity EGFR ligands selected from betacellulin, EGF,HB-EGF or TGFα in the biopsy sample, and c) comparing the average levelof protein or mRNAs coding for each of the high affinity EGFR ligandsmeasured in a) to the average level of protein or mRNAs coding for eachof the low affinity EGFR ligands measured in b); wherein, if the averagelevel of protein or mRNAs coding for low affinity EGFR ligands measuredin a) is greater than the average level of protein or mRNAs coding forhigh affinity EGFR ligands measured in b), the patient is predicted tohave the favorable outcome as a result of treatment #1 and the patientis also predicted to have the favorable outcome as a result of treatment#2, and if the average level of protein or mRNAs coding for low affinityEGFR ligands measured in a) is less than or equal to the average levelof protein or mRNAs coding for high affinity EGFR ligands measured inb), the patient is predicted to have an unfavorable outcome fromtreatment #1 and is predicted to have a favorable outcome from treatment#2.